PROJECT SUMMARY The overall goals of this project are to develop powerful technology that efficiently generates high-performance and renewable antibodies to post-translational modifications (PTMs), and to make such reagents broadly available to the research community. PTMs are chemical modifications of proteins that are important in many cellular functions. Dysregulation of PTMs contributes to many diseases, including cancer. Antibodies to PTMs are a central component in analyzing PTMs, but many available antibodies have severe shortcomings, limiting the progress of biomedical and cancer research. Two major issues with available antibodies are low quality and lot-to-lot variation, which could lead researchers to incorrect conclusions and contribute to a lack of reproducibility in research results. Moreover, recent advances in proteomics and genomics have enabled comprehensive studies that produce large datasets, and the community shares those results. Thus, the antibody problem has become a world-wide problem affecting diverse research fields. The generation of antibodies with high specificity and high affinity to PTMs is challenging, because they must discriminate small chemical changes in amino acids and closely related amino acid sequences. To overcome fundamental difficulties in molecular recognition, we propose an innovative approach built on our previous discovery of a unique antigen-binding mode of high- performance antibodies to histone methylation. Conventionally, the antigen-binding fragment (Fab) of an antibody recognizes its antigen with 1:1 stoichiometry. Our previous studies of antibodies to histone methylation revealed an unexpected binding mechanism, which we dubbed ?antigen clasping?, where two Fabs cooperatively clasp one antigen by forming head-to-head homodimers. Antigen clasping creates exceptionally large antigen- recognition surfaces, which enables antibodies to achieve high specificity and high affinity to PTMs. We hypothesize that an approach to rationally generate antibodies that use antigen clasping will substantially accelerate the development of high-performance antibodies to PTMs. Our specific aims are to establish a rational approach for generating clasping antibodies, and to demonstrate the broad applicability of our approach by generating clasping antibodies to phosphorylated antigens. We will critically validate clasping antibodies and benchmark them against available antibodies. Primary products of this project will be recombinant proteins with defined sequences, eliminating a major barrier to reproducibility. We envision that the proposed technology and the high-performance reagents it produces will enable more robust and thorough analyses of PTMs and their roles in diseases such as cancer.